HLA-DRB1*15:03 and HLA-DRB1*11: useful predictive alleles for alloantibody production in thalassemia patients.

BACKGROUND AND OBJECTIVES: Recently, thalassemia has been introduced as a chronic disease. In spite of prolonging life in thalassemia patients, the quality of their life has not significantly improved. One of the challenges that makes their quality of life poor is alloimmunisation which causes several complications to patients by restricting their options. Some individuals are more susceptible to developing an alloantibody than others. They are categorised as responders and non-responders. Determining responders before the first transfusion allows transfusion services to provide compatible blood and prevent alloimmunisation. The aim of the present study was to determine the relationship between HLA-DRB1*15:03, HLA-DRB1*11 and HLA-DRB1*09:01 alleles and alloimmunisation in Iranian thalassemia patients. MATERIALS AND METHODS: Antibody screening tests were performed by tube method, and HLA-DRB1 genotyping was determined by Sequence-Specific Primers (SSP-PCR) in 59 alloimmunised and 205 non-alloimmunised patients. HLA-DRB1 allele frequencies were compared between alloantibody-positive and -negative groups through the χ2 test. RESULTS: HLA-DRB1*15:03 allele frequency was significantly different between groups (P = 0·000, odds ratio (OR) = 4·193). There was a correlation between HLA-DRB1*11 and anti-K (P = 0·000, OR = 6·643). There was no association between HLA-DRB1*09:01 and alloimmunisation (P = 0·350). CONCLUSIONS: According to our results, detecting HLA-DRB1*15:03 and HLA-DRB1*11 alleles are useful in the pre-transfusion test and could determine responder patients and improve transfusion safety.

The rs2910164 variant is associated with reduced miR-146a expression but not cytokine levels in patients with type 2 diabetes.

PURPOSE: Previous reports have demonstrated that genetic variations in microRNAs regulome could affect microRNAs-mediated regulation. Therefore, in the present study we were aimed at (1) comparison of microRNA 146-a (miR-146a) peripheral blood mononuclear cells (PBMCs) and plasma levels between diabetic patients and controls, and (2) investigating the possible association of rs2910164 with miR-146a and its related target genes expression and also serum cytokine levels. METHODS: The study population consisted of 60 subjects including 30 type 2 diabetes (T2D) patients and 30 controls with determined genotypes for rs2910164. The RNA expression levels were determined by real-time PCR. Moreover, TNF-α, IL-6, IL-10 and IL-1ß serum levels were measured using ELISA method. RESULTS: Our results showed that the miR-146a expression levels were significantly decreased in PBMCs (P = 0.004) and plasma (P = 0.008) samples of patients with T2D compared to healthy participants. In addition, we observed that IRAK1 mRNA expression-but not TLR4, TRAF6 and NFĸB-was significantly increased in patients with T2D compared to controls (P = 0.028). The relative expression levels of miR-146a in plasma and PBMCs samples of diabetic patients with the rs2910164 GG genotypes were significantly higher than that in CC (P < 0.05). Moreover, no significant differences were found in miR-146a targets and cytokine levels between the rs2910164 different genotypes. CONCLUSION: Our study demonstrated that miR-146a circulating levels were significantly elevated in controls compared with T2D patients. In addition, we identified that rs2910164-C allele is associated with reduced expression levels of the miR-146a but not its mRNAs targets and cytokine levels in diabetic patients.

DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice.

Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis. The aim of the present study was to compare the immune responses induced following DNA vaccination with LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genes alone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immune responses were evaluated before and after challenge with Leishmania major (L. major). In addition, the mean lesion size was also measured from 3th week post-infection. All immunized mice showed a partial immunity characterized by higher interferon (IFN)-γ and Immunoglobulin G (IgG2a) levels compared to control groups (p<0.05). IFN-γ/ Interleukin (IL)-4 and IgG2a/IgG1 ratios demonstrated the highest IFN-γ and IgG2a levels in the group receiving LACK-TSA fusion. Mean lesion sizes reduced significantly in all immunized mice compared with control groups at 7th week post-infection (p<0.05). In addition, there was a significant reduction in mean lesion size of LACK-TSA and TSA groups than LACK group after challenge (p<0.05). In the present study, DNA immunization promoted Th1 immune response and confirmed the previous observations on immunogenicity of LACK and TSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccine can induce stronger immune responses and protection against infectious challenge with L. major.

Vitronectin and Urokinase-Type Plasminogen Activator Gene Expression Levels Are Increased in Patients with Coronary Artery In-Stent Restenosis.

Neointimal hyperplasia is known as a main factor contributing to in-stent restenosis (ISR). Monocytes may play a central role in vessel restenosis process after stent implantation. The aim of this study was to investigate the relationships between the urokinase-type plasminogen activator (PLAU) and vitronectin (Vtn) gene expression levels in peripheral blood mononuclear cell samples isolated from whole blood of 66 patients undergoing coronary artery angiography (22 controls, stenosis < 0.05%; 22 with stent no-restenosis and stenosis < 70%; and 22 with ISR and stenosis > 70%). The Vtn and PLAU gene expression levels were measured by real-time quantitative polymerase chain reaction technique. The age- and gender-independent increases in the expression levels of Vtn (17-fold; p < 0.001) and PLAU (27-fold; p < 0.0001) genes were found in the patients with ISR as compared with the control group. The results suggested that the Vtn and PLAU genes may be involved in the coronary artery ISR.

Quantification of Active and Latent Form of Human Cytomegalovirus Infection in Umbilical Cord Blood Donors by Real-Time PCR.

BACKGROUND: Umbilical cord blood (UCB) is believed to be a highly valuable source of hematopoietic stem cells for transplantation. Objective: To investigate the prevalence of active and latent human cytomegalovirus (CMV) infection in UCB donors in Iranian population. METHODS: A total of 825 UCB samples was collected under standard procedures and analyzed for the presence of CMV DNAs in buffy coat (latent infection) and plasma (active infection). DNA was extracted from buffy coat and plasma samples separately and tested with quantitative real-time PCR. All positive samples were checked by ELISA for IgG and IgM anti-CMV antibody. RESULTS: Latent CMV infection was detected in 17 (2%) buffy coat samples with a low level of viral load, which indicated the presence of latent viral infection in donors. None of the plasma samples were found positive for CMV DNA reflecting no active infection. In the 17 positive samples, CMV viral load was 91-104 (mean: 100) copies/mL. All samples positive for viral DNA were also found positive for CMV IgG antibody by ELISA. No CMV IgM antibody was detected in positive samples. CONCLUSION: CMV is still the most important virus that infects hematopoietic stem cells and could be dangerous, especially for immunocompromized transplant recipients. We therefore suggest using real-time PCR for the detection and quantification of the viral DNA in buffy coat and plasma of UCB donors. PCR of plasma for detection of CMV and antibody assay for CMV infection add no more sensitivity for the detection of latent CMV infection in UCB donors.

Synthesis, characterization and biological evaluation of novel α, ß unsaturated amides.

Three derivatives of α,ß unsaturated amides have been successfully synthesized via Ugi-four component (U-4CR) reaction. The interactions of the amides with calf thymus deoxyribonucleic acid (ct-DNA) have been investigated in the Tris-HCl buffer (pH=7.4) using viscometric, spectroscopic, thermal denaturation studies, and also molecular docking. By UV-Vis absorption spectroscopy studies, adding CT-DNA to the compound solution caused the hypochromism indicates that there are interactions between the compounds and DNA base pairs. In competitive fluorescence with methylene blue as an intercalator probe, adding compounds to DNA-MB solution caused an increase in emission spectra of the complex. This could be because of compound replacing, with similar binding mode of MB, between the DNA base pairs due to release of bonded MB molecules from DNA-MB complex. Thermal denaturation studies and viscometric experiments also indicated that all three investigated compounds bind to CT-DNA by non-classical intercalation mode. Additionally, molecular docking technique predicted partial intercalation binding mode for the compounds. Also, the highest binding energy was obtained for compound 5a. These results are in agreement with results obtained by empirical methods.

Codelivery of DNA vaccination encoding LeIF gene and IL-12 increases protection against Leishmania major infection in BALB/c mice.

Previous studies have shown that Leishmania elongation initiation factor (LeIF) antigen causes a partial immunity against leishmaniasis. The antigen develops type I immunity by overexpression of inflammatory cytokines such as interleukin-12 (IL-12), IFN-γ and TNF-α. Therefore, We evaluated immune responses induced by the LeIF gene against Leishmania major infection. Immunization with LeIF gene alone or with IL-12 induced Th1 response and produced higher IFN-γ and lower IL-4 levels by splenocytes than control groups (P < 0·05) and also ratios of IFN-γ/IL-4 were 11·68 to 18·53 times more in immunized groups than control groups after challenge. In addition, analysis of humoral immune response revealed that immunized mice had more IgG2a levels than both control groups (P < 0·05). On the other hand, lesion size was less for immunized animals than control groups from 4th week after challenge (P < 0·05). The percentage reduction in lesion size was 29·30% for LeIF and 51·98% for LeIF + IL-12 than PBS at 12th week post-infection. Spleen parasite burden decreased in all immunized groups in comparison with control groups (P < 0·05). The results indicated that LeIF gene induced partial immunity against L. major infection in BALB/c mice. However, LeIF plus IL-12 group showed more potent immunity with smaller lesions than other groups.

Weight management behaviors in a sample of Iranian adolescent girls.

PURPOSE: Attempts to obtain the ideal body shape portrayed in advertising can result in behaviors that lead to an unhealthy reduction in weight. This study was designed to identify contributing factors that may be effective in changing the behavior of a sample of Iranian adolescents. METHODS: Three hundred fifty adolescent girls from high schools in Kerman, Iran participated in a cross-sectional study based on a self-administered questionnaire. Multifactorial logistic regression modeling was used to identify the factors influencing each of the contributing factors for body management methods, and a decision tree model was constructed to identify individuals who were more or less likely to change their body shape. RESULTS: Approximately one-third of the adolescent girls had attempted dieting, and 37 % of them had exercised to lose weight. The logistic regression model showed that pressure from their mother and the media; father's education level; and body mass index (BMI) were important factors in dieting. BMI and perceived pressure from the media were risk factors for attempting exercise. CONCLUSIONS: BMI and perceived pressure from relatives, particularly mothers, and the media were important factors in attempts by adolescent girls to lose weight.

Cloning and Constructing a Plasmid Encoding Leishmania Eukaryotic Initiation Factor Gene of Leishmania major Fused with Green Fluorescent Protein Gene as a Vaccine Candidate.

OBJECTIVE: Leishmaniasis is usually treated with chemotherapy; however, toxicity, resistance and high-cost limit use of the chemical drugs. Leishmania eukaryotic initiation factor (LeIF) protein acts the same as interleukin (IL)-12 and reduces the secretion of IL-4 in lymph node cells of mice infected with Leishmania major. The aim of this study was cloning of the gene encoding LeIF antigen into eukaryotic expression plasmid pEGFP-N1. METHODS: DNA was extracted from Iranian strain of the L major (MRHO/IR/75/ER) promastigotes. The full-length sequence of LeIF was amplified with Pfu DNA polymerase using a specific primer. The amplified LeIF was cloned into a pJET1.2/blunt vector. Then this fragment was digested with HindIII and EcoRI and was subcloned into the pEGFP-N1 vector. Confirmation of the cloning was done by colony polymerase chain reaction (PCR). RESULTS: Leishmania eukaryotic initiation factor gene was successfully cloned and subcloned into pJET1.2 and pEGFP-N1 plasmids, respectively. The results of colony PCR, restriction analysis and sequencing confirmed them. CONCLUSIONS: We cloned LeIF gene which could be expressed in eukaryotic cells in vivo and could be used as a vaccine candidate against leishmaniasis in future studies.

Quantitative polymerase chain reaction for detection of human herpesvirus-7 infection in umbilical cord blood donors.

OBJECTIVE: Umbilical cord blood (UCB) has been a reasonable alternative to granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow, as a source of hematopoietic stem cells with a lower risk of graft-versus-host disease. In immunocompromised hosts after transplantation, the risk of viral infection in adults, especially with beta-herpesviruses such as human herpesvirus-7 (HHV-7), may be increased. This virus in immunocompromised patients can be reactivated from latency and converted to an active phase. Therefore, light-upon-extension real-time polymerase chain reaction (PCR) was developed to assess the prevalence and load of HHV-7 in the plasma and buffy coat of donors. METHODS: About 825 UCB samples under standard protocol from donors were collected. Then, DNA from plasma and buffy coat was extracted and quantitative real-time PCR was performed with light-upon-extension primers. RESULTS: Overall, HHV-7 was detected in 3.64% (30/825) of UCB donors. HHV-7 DNA was detected in 26 (3.2%) buffy coat samples (latent infection), and only 4 (0.48%) of them were positive for HHV-7 DNA in plasma samples (active infection); the mean HHV-7 viral load was 1.31 × 10(1) copies/mL in latent infection, and 1.94 × 10(5) copies/mL in active infection. CONCLUSIONS: We suggest that real-time PCR in plasma and buffy coat could be a useful method to detect active and latent HHV-7 infection in UCB donors and determine its role in subsequent transmission events.

Methylene blue based device for pathogen reduction in human plasma.

BACKGROUND: Despite improvement in safety of plasma transfusion some virus transmission still remains a problem. So as World Health Organization (WHO) recommends, many countries developed Pathogen Reduction Technologies (PRT) to inactivate pathogens, in plasma components. The Methylene Blue (MB) based methods is one of the most universal one. The purpose of this research was, produce a device that can inactivate viruses in MB environment. MATERIALS AND METHODS: In this interventional study, each Plasma Sample was illuminated by 70Pieces (PCs) of 1 w red Light Emitting Diodes (LEDs) from one side. These LEDs emit light at central wavelength of 627 nm with 20 nm Full Width at Half Maximum (FWHM). Two model viruses Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were used and Tissue Culture 50% Infection Dose (TCID50) was used to calculate virus Log reduction. Two concentration of MB and 5 different illumination times were used. RESULTS: In 10 µm concentration of MB, HSV had 6.00±0.2 maximum log reduction that obtain after 60 minutes illumination and VSV had 5.50± 0.3 maximum log reduction after 75 minutes illumination. In 1 µM concentration of MB, HSV had 5.20±0.3 maximum log reduction that obtain after 60 minutes illumination and VSV had 4.90± 0.2 maximum log reduction after 75 minutes illumination. CONCLUSION: Results of virus inactivation in this method were similar to other methods (P-value<0.05 in comparison with Spring method, and P-value>0.05 in comparison with Theraflex), and it showed this device could inactivate viruses according to WHO recommendation.

Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis.

Toxoplasma gondii, the pathogen of toxoplasmosis, can infect most mammals and birds. The high incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. We constructed a DNA cocktail, containing plasmids encoding the full-length SAG1 and ROP2 genes of T. gondii and evaluated its immune response and protective efficacy in comparison with single-gene vaccines and control groups. We immunized BALB/c mice intramuscularly three times. DNA cocktail elicited IgG and IFN-γ, TNF-α and IL-2 greater than single-gene plasmids and increased survival time against a lethal challenge with the highly virulent T. gondii RH strain. The current study shows that pc-SAG1+ pc-ROP2 as a cocktail DNA vaccine produces higher Th1 immune response than single-gene plasmids and cocktail DNA is effective to prime an enhanced and balanced specific immunity.

The Effect of Vitamin D3 Alone and Mixed With IFN-γ on Tachyzoites of Toxoplasma gondii (RH Strain) Proliferation and Nitric Oxide (NO) Production in Infected Macrophages of BALB/C Mice.

INTRODUCTION: Toxoplasma gondii is an obligatory interacelullar parasite that infects nucleated cells in its intermediate hosts. The aim of the present study was to determine the effect of vitamin D3 on the multiplication of T. gondii in peritoneal macrophage of Balb/c mice and nitric oxide production by macrophages. METHODS: According to usage of vitamin D3 (one dose or seven doses) and INFγ in vitro and in vivo, this study was divided into four experiments. In all experiments, the macrophages were collected from peritoneum and cultured in RPMI-1640. Then the supernatants were collected after 24 h and their nitric oxide was measure. After 96 h, the macrophages were collected and stained and the number of tachyzoites was measured. RESULTS: The first experiment (the mice were infected with tachyzoites and after 2 h, got one dose vitamin D3 intraperitonealy) showed the best results. The mean of tachyzoites per macrophages was 2.37, and mean±SD of nitric oxide was 187.8±9. DISCUSSION: High-level production of nitric oxide may be related to the only one injection of vitamin D3. The injection in long time might suppress the immune system.

Inactivation of virus in intravenous immunoglobulin G using solvent/detergent treatment and pasteurization.

The safety of plasma derived medicinal products, such as immunoglobulin, depends on viral inactivation steps that are incorporated into the production process. Several attempts have been made to validate the effectiveness of these inactivation methods against a range of physio-chemically diverse viruses. Treatment with solvent/detergent (S/D) and pasteurization (P) has been continuously used in our IgG production and these methods were analysed in this study as models of viral inactivation. Bovine Viral Diarrhoea Virus (BVDV), Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were employed as models of HCV, HBV and HIV respectively. Polio and Reo viruses also were used as stable viruses to chemical substances. The infectivity of a range of viruses before and after treatment with two methods of viral inactivation was measured by end point titration and their effectiveness expressed as Logarithmic Reduction Factors (LRF). Solvent/detergent treatment reduced the amount of enveloped viruses by 5-6 logs. The reduction factor was between 5-6 logs for all viruses used in the pasteurization process. A final log reduction factor was obtained as the sum of the two individual methods. Both inactivation methods have advantages and disadvantages with respect to their ability to inactivate viruses. Thus,combination of two robust virus inactivation steps, solvent/detergent and pasteurization, increases the safety margin of immunoglobulin preparations.